Fig. 8

Over-expression of HSV-1 gD and HIV-1 Env still results in exclusion of HIV gp120/gp41 from particles. 293 cells were co-transfected with either the empty pcDNA3.1(+) vector and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or vectors expressing gD, Bal Env and pNL4-3. At 30 h post-transfection, the cells were starved for 2 h with methionine/cysteine-free media and then radiolabeled for 16 h with ³⁵S-methionine/cysteine. The culture media was harvested and subjected to low speed centrifugation to remove cellular debris. The resulting supernatant was layered on a 20% sucrose cushion and subjected to ultracentrifugation to pellet viral particle as described in the Materials and methods. The pelleted virus was harvested, resuspended in 200 μl of DMEM without serum and layered on a discontinuous 20-60% sucrose gradient. The virus was subjected to ultracentrifugation for 20 h at 76,000×g in a SW55Ti at which time the fractions were collected and proteins immunoprecipitated using anti-HIV-1 antibodies to immunoprecipitate HIV-1 Gag and Env or a monoclonal antibody to immunoprecipitate HSV-1 gD. The immunoprecipitates of the HIV-1 proteins and HSV-1 gD from gradient fractions of cells co-transfected with pcDNA3.1(+) and pNL4-3 or gD and pNL4-3 were the same as in Fig. 7. a Immunoprecipitation of HIV-1 proteins and HSV-1 gD from cell lysates of cells co-transfected with pcDNA3.1(+) and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gD, Bal Env and pNL4-3. b Immunoprecipitation of HIV-1 proteins from supernatants prior to and after ultracentrifugation through a 20% sucrose cushion from cells co-transfected with pcDNA3.1 and pNL4-3, a vector expressing Bal Env and pNL4-3, a vector expressing gD and pNL4-3, or a vector expressing gD and Bal Env and pNL-3. c Immunoprecipitation of HSV-1 gD from viral pellet after ultracentrifugation through a 20% sucrose cushion from cells transfected with pcDNA3.1(+) and pNL4-3, a vector expressing gD and pNL4-3, a vector expressing Bal Env and pNL4-3, or a vector expressing gD, Bal Env, and pNL4-3. d Immunoprecipitation of HIV-1 proteins from gradient fractions of cells co-transfected with Bal Env and pNL4-3. e, f Immunoprecipitation of HIV-1 proteins (e) and HSV-1 gD (f) from gradient fractions of cells co-transfected with a vector expressing gD, Bal Env, and pNL4-3